Two-dimensional electrophoresis is widely used to separate complex protein mixtures. By permitting separation to occur on the basis of two different sets of properties in succession, two-dimensional electrophoresis provides much higher resolving power than that obtainable in a single stage separation.
The technique has been used to combine separation parameters in a variety of ways. Separation based on charge for example has been performed in the first stage, followed by separation based on molecular weight in the second. Likewise, separation in one gel concentration has been followed by separation in another concentration of the same gel. As further examples, two stages have been used to effect a stepwise change in pH, a homogeneous gel followed by a pore gradient gel, the use of two different protein solubilizers or two concentrations of the same solubilizer, a discontinuous buffer system in one stage and a continuous buffer system in the other, and the use of isoelectric focusing followed by homogeneous or pore gradient electrophoresis. Such techniques have permitted the separation of serum or cell proteins, bacterial proteins, non-histone chromatin proteins, ribosomal proteins, mixtures of ribonucleoproteins and ribosomal proteins, nucleic acids, and similar materials.
The basic procedure in a two-dimensional system begins with a first dimension separation in an elongate or rod-shaped gel, such as one having a diameter on the order of 5.0 mm, with migration and separation occurring along the gel axis until the solutes or proteins are distributed among zones positioned along the length of the rod. This is followed by placement of the rod along one edge of a slab gel to effect the second dimension--migration of the solutes from each zone into the slab gel in the direction transverse to the rod gel axis.
The difficulties encountered in procedures of this type are those relating to the transfer of the rod-shaped gels after the first dimension separation has occurred to prepare for the second dimension separation. The first dimension separation generally takes place in the tube in which the rod gel is originally cast. Once the first dimension is complete, the gel rod with the solute zones is removed from the tube by physical means such as extrusion, then placed along the exposed edge of the slab gel. These manipulations require delicate handling, and even with the exercise of a great deal of care, there remains the risk of damage to the gel and distortion or disturbance of the solute zones. Once extracted, the rod gel must be properly aligned with the slab gel, and placed in full contact for purposes of both electrical continuity and unobstructed solute migration between the gels. These are further sources of error and lack of reproducibility. In addition to the handling difficulties and potential for inaccuracies and lack of reproducibility, the time required in the handling and placement of the rod gel is detrimental to operator efficiency, and any fatal errors will result in irretrievably lost time and data. Some have used gel strips for the first dimension separation, but similar difficulties, errors and lack or reproducibility have been encountered.
The present invention resides in a pre-cast twodimensional gel system, with the first and second dimension gels combined on a single gel support in which both stages of the separation take place. The support may be an enclosure comprising a single pair of plates, with appropriate spacers to set the spacing between the plates and define the gel thicknesses. Both first and second dimension gels are cast and retained between the plates. The second dimension gel is a slab occupying a portion of the space between the plates, while the first dimension is a strip positioned in the remaining space, parallel to the slab, with a removable electrically insulating layer separating the gels. Alternatively, the support may be gel backing comprised of a sheet of plastic, gel bond or other supporting material on which both first and second dimension gels are cast, with the insulating layer, which may be a layer of insulating material or an air space, between them.
The first dimension separation is thus performed by loading the sample on one end of the strip gel and imposing an electric field across the strip gel in the lengthwise direction while the two gels are electrically insulated from each other, then removing the electrically insulating layer and placing the two gels in electrical contact along their full length, followed by imposing an electric field across both in a direction transverse to the first. The electrically insulating layer may be solid, liquid or gas, and electrical contact between the two gels for the second dimension separation maybe achieved either by direct contact or by the insertion of an electrically conductive layer. The invention eliminates the need for transferring a rod or strip gel from one support enclosure to another between the two stages of the separation, thereby lessening the risk of gel damage and zone distortion and lowering the incidence of inaccuracies and systematic error.
Further objects, advantages and features of the invention will be apparent from the description which follows.